Synthesis and Evaluation of Manganese(II)-Based Ethylenediaminetetraacetic Acid-Ethoxybenzyl Conjugate as a Highly Stable Hepatobiliary Magnetic Resonance Imaging Contrast Agent

Article abstract of DOI:10.1021/acs.bioconjchem.8b00560

In this study, we designed and synthesized a highly stable manganese (Mn²⁺)-based hepatobiliary complex by tethering an ethoxybenzyl (EOB) moiety with an ethylenediaminetetraacetic acid (EDTA) coordination cage as an alternative to the well-established hepatobiliary gadolinium (Gd³⁺) chelates and evaluated its usage as a T₁ hepatobiliary magnetic resonance imaging (MRI) contrast agent (CA). This new complex exhibits higher r₁ relaxivity (2.3 mM^-1 s^-1) than clinically approved Mn²⁺-based hepatobiliary complex Mn-DPDP (1.6 mM^-1 s^-1) at 1.5 T. Mn-EDTA-EOB shows much higher kinetic inertness than that of clinically approved Gd³⁺-based hepatobiliary MRI CAs, such as Gd-DTPA-EOB and Gd-BOPTA. In addition, in vivo biodistribution and MRI enhancement patterns of this new Mn²⁺ chelate are comparable to those of Gd³⁺-based hepatobiliary MRI CAs. The diagnostic efficacy of the new complex was demonstrated by its enhanced tumor detection sensitivity in a liver cancer model using in vivo MRI.

Full text of DOI:10.1021/acs.bioconjchem.8b00560

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Article
Synthesis and Evaluation of Manganese (II)-based
Ethylenediaminetetraacetic Acid-Ethoxybenzyl Conjugate as a Highly
Stable Hepatobiliary Magnetic Resonance Imaging Contrast Agent
Md. Kamrul Islam, Soyeon Kim, Hee-Kyung Kim, Yeoun-Hee Kim, Young-Mi Lee,
Garam Choi, Ah Rum Baek, Bo Kyung Sung, Minsup Kim, Art E. Cho, Hyo Jeung
Kang, Gang-Ho Lee, Seon Hee Choi, Taekwan Lee, Ji Ae Park, and Yongmin Chang
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00560 • Publication Date (Web): 01 Nov 2018
Downloaded from http://pubs.acs.org on November 2, 2018
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Bioconjugate Chemistry
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Synthesis
and
Evaluation
of
Manganese
(II)-based
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Ethylenediaminetetraacetic Acid-Ethoxybenzyl Conjugate as a
Highly Stable Hepatobiliary Magnetic Resonance Imaging Contrast
Agent
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Md. Kamrul Islam,a,† Soyeon Kim,a,† Hee-Kyung Kim,b,c Yeoun-Hee Kim,d Young-
Mi Lee,c Garam Choi,a Ah Rum Baek,a Bo Kyung Sung,a Minsup Kim,e Art E.
Cho,e Hyojeung Kang,f Gang-Ho Lee,g Seon Hee Choi,h Taekwan Lee,h Ji-Ae
Park,i and Yongmin Changj,k,*
f
g
aDepartment of Medical & Biological Engineering, Department of Pharmacy, Department of
Chemistry, Kyungpook National University, 80, Daehak-ro, Buk-gu, Daegu 41566, Korea
c
bBK21 Plus KNU Biomedical Convergence Program, School of Medicine, Institute of
j
Biomedical Engineering Research, Department of Molecular Medicine, School of Medicine,
Kyungpook National University, 680, Gukchaebosang-ro, Jung-gu, Daegu 41944, Korea
dInstitute of New Drug Research, Myungmoon Bio, 180, Yuram-ro, Dong-gu, Daegu, 41059,
Korea.
eDepartment of Bioinformatics, Korea University Sejong Campus, 2511, Sejong-ro, Sejong city,
30019, Korea.
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hLaboratory Animal Center, Daegu-Gyeongbuk Medical Innovation Foundation,
80, Chumbok-ro, Dong-gu, Daegu 41061, Korea
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iMolecular Imaging Research Center, Korea Institute of Radiological and Medical Sciences,
Seoul 139-706, Korea
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kDepartment of Radiology, Kyungpook National University Hospital, 130 Dongdeok-ro, Jung-
gu, Daegu 41944, Korea
† These authors contributed equally to this article.
* Corresponding author: E-mail: ychang@knu.ac.kr
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ABSTRACT
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In this study, we designed and synthesized a highly stable manganese (Mn2+)-based hepatobiliary
complex by tethering an ethoxybenzyl (EOB) moiety with an ethylenediaminetetraacetic acid
(EDTA) coordination cage as an alternative to the well-established hepatobiliary gadolinium
(Gd3+) chelates and evaluated its usage as a T1 hepatobiliary magnetic resonance imaging (MRI)
contrast agent (CA). This new complex exhibits higher r1 relaxivity (2.3 mM−1 s−1) than
clinically approved Mn2+-based hepatobiliary complex Mn-DPDP (1.6 mM−1 s−1) at 1.5 T. Mn-
EDTA-EOB shows much higher kinetic inertness than that of clinically approved Gd3+-based
hepatobiliary MRI CAs, such as Gd-DTPA-EOB and Gd-BOPTA. In addition, in vivo
biodistribution and MRI enhancement patterns of this new Mn2+ chelate are comparable to those
of Gd3+-based hepatobiliary MRI CAs. The diagnostic efficacy of the new complex was
demonstrated by its enhanced tumor detection sensitivity in a liver cancer model using in vivo
MRI.
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INTRODUCTION
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Hepatocellular carcinoma (HCC) is currently one of the leading causes of cancer related death,
which represents over 90% of all primary liver cancers.1 If diagnosis of the HCC is late,
malignancy has an unfavorable prognosis. This year, it is estimated that 30,200 deaths (20,540
men and 9,660 women) will occur in the United States by this disease.2 Early diagnosis and
effective treatment can increase the long-term survival rate up to 50%–70% (5-year survival).3
Thus, liver-specific CAs are important for early diagnosis of the HCC.
Gadolinium (Gd3+) is a popular choice amongst paramagnetic metals used as contrast agents
(CAs) for magnetic resonance imaging (MRI) owing to its suitable magnetic properties.4-6
However, some Gd3+-based imaging probes are associated with the initiation of nephrogenic
systemic fibrosis (NSF), a rare but fatal fibrosis of the organs that can arise in patients with
compromised renal function.7,8 Furthermore, a number of reports demonstrated that
intravenously administered Gd3+-based imaging agents accumulate in the brains of patients with
normal renal function, further questioning the use of certain marketed Gd3+-based CAs.9-11
Therefore, non-lanthanide metals, particularly less toxic and more biofriendly Mn2+-based
complexes, are receiving special attention for the development of next-generation potent MRI-
CAs.12-17
Mn2+ ions with spin number (S = 5/2) possess fast water exchange kinetics and slow
longitudinal electronic relaxation, which are necessary physical properties for MR CAs.18-21
However, Mn2+ ions are not free from in vivo toxicity. The use of Mn2+ ions poses toxicological
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concerns because overexposure to this metal ion can lead to progressive and permanent damage
to the brain.22 Although small amounts of Mn2+ are required by the human body, overexposure to
free Mn2+ ions may cause neurotoxicity that can develop into a Parkinson-like disease called
manganism.23 In addition, cardiovascular, liver, as well as reproductive and developmental
toxicities have been reported. The typical symptoms of manganism often correlate with the
degree of Mn2+ ion accumulation in the globus pallidus, which is seen as hyper intensity on a T1
MRI.24 Mn-DPDP (mangafodipir trisodium; Chart 1), approved by the food and drug
administration (FDA) for use as a liver imaging agent, releases free Mn2+ ions in the blood vessel
after intravenous (IV) administration, which are responsible for T1 contrast enhancement.25 Mn-
DPDP showed relatively low relaxivity; thus, an increased dose is required to generate sufficient
contrast enhancement, and it may lead to unexpected toxic side effects. Although Mn-DPDP is
the first liver-specific hepatobiliary MR CA, it is no longer available for clinical use because of
low relaxivity and potential safety issues.26 Therefore, even for Mn2+-based MRI-CAs, the in
vivo stability of the complex is very important to minimize potential toxicity. Because Mn2+ is
essential for mitochondrial function and hepatocytes are mitochondria-rich liver cells, Mn2+
homeostasis in the liver is very important and all Mn2+-based liver-specific hepatobiliary MRI-
CAs require high in vivo stability so as to not release free Mn2+.
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Numerous efforts have been made to design stable Mn2+-based complexes with reduced
toxicity for in vivo MRI applications. Zheng et al synthesized three nanoglobular macrocyclic
Mn(II) chelate conjugates for use as promising non-Gd MRI CAs.27 Recently, a fibrin-targeted
-
[Mn(PyC3A) (H2O)] complex was synthesized by Caravan et al. as a molecular agent for
vascular imaging, and it showed high stability at pH 7.4 (log KML = 11.40).28 More recently, we
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reported a novel Mn(II) chelate of ethylenediaminetetraacetic acid-benzothiazole aniline (EDTA-
BTA) conjugate (Chart 1) as a liver-specific agent based on the lipophilic character of BTA
moiety, and it showed comparable kinetic inertness to Gd3+-based agents.29 Compared to Mn-
EDTA-BTA, Mn-EDTA-EOB showed high kinetic stability and this high stability will reduce
brain accumulation of free manganese ion and thus will reduce possible neurotoxicity such as
manganism. Therefore, the design of highly stable hepatobiliary Mn2+ complexes still remains a
challenge.
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In the current study, we introduce a new chelator EDTA-ethoxybenzyl (EOB) (L) that forms a
highly inert complex with Mn2+. We designed the highly stable Mn2+ chelate based on an EDTA
coordination cage bearing an EOB moiety for use as a hepatobiliary agent. EOB is a lipophilic
moiety, which was used in the clinically approved Gd-based hepatobiliary agent, Gd-DTPA-
EOB (gadolinium ethoxybenzyl diethylenetriamine pentaacetic acid; Chart 1) chelate.30 Gd-
DTPA-EOB showed 50% accumulation in functioning hepatocytes via excretion into the biliary
tract and 50% through the kidneys.26 In the present study, we aimed to design a complex with
Mn2+ ions, which has higher chelation stability than Gd3+-based liver-specific MRI CAs, such as
Gd-DTPA-EOB and Gd-BOPTA. In addition, we expected that the newly synthesized small
Mn2+ chelate-based hepatobiliary agent showed much higher in vivo stability than the recent Mn
(II) chelate of EDTA-BTA conjugate. Moreover, computational modeling studies were
performed to predict the possible molecular mechanism of hepatocyte uptake of newly
synthesized Mn-EDTA-EOB. We demonstrated that this new hepatobiliary Mn chelate is rapidly
taken up by liver hepatocytes and excreted through biliary and renal pathways, and is highly
promising for clinical liver imaging applications such as liver cancer imaging.
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Chart 1. Structure of the compounds discussed in this study.
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S
N
HN
N
2-
OPO3 2-O3PO
O
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O
O
O
O
N
O
N
O
N
O
N
O
N
O
N
N
Mn
Mn
O
O
O
O
Mn
O
O
O
O
O
O
O
O
O
Mn-DPDP
Mn-EDTA
Mn-EDTA-BTA
O
O
O
O
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O
O
N
O
O
N
O
N
N
Gd
N
O
O
O
O
O
O
N
N
O
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N
O
Gd
Gd
O
O
O
O
O
O
O
O
O
O
O
O
O
O
Gd-DTPA-EOB
Gd-BOPTA
Gd-DTPA
RESULTS AND DISCUSSION
Synthesis and characterization. Scheme 1 shows the procedure for the synthesis of the chelate
conjugate and its Mn2+ complex (abbreviated as L and Mn-EDTA-EOB, correspondingly). The
synthesis of Mn-EDTA-EOB was started from D,L-2,3-diaminopropionic acid
monohydrobromide. Compound 1 was synthesized according to a previously published study,29
and then conjugated with 4-ethoxyaniline in the presence of O-(benzotriazol-1-yl)-N, N, N′, N′-
tetramethyluronium hexafluorophosphate (HBTU) to form compound 2 as a white solid. A
mixture of trifluoroacetic acid (TFA) and dichloromethane (CH2Cl2) was used to cleave tert-
butyl ester and a pale-yellow product 3 was obtained after precipitation. Alkylation with tert-
butyl bromoacetate under previously reported conditions resulted in a high yield of the protected
EDTA-EOB ligand 4.31 Deprotection of the tert-butyl group was accomplished using TFA, and
the pure ligand L was collected from the reaction mixture as an off-white solid with 53% overall
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yield. The Mn2+ complex was synthesized by reaction of ligand L with MnCl2·4H2O (1 equal) at
pH 6.5, and the inorganic impurities were removed by using reverse-phase column
chromatography. Lyophilization was used to isolate the pure chelate as a sodium salt with 31%
overall yield. The ligand formation and its Mn2+ complex was evaluated by microanalysis and
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1
spectroscopic methods, such as H NMR, HR-FAB-MS, ESI-MS, and elemental analysis (see
Supporting Information).
Scheme 1. Synthesis of Mn-EDTA-EOB complex. a
O
O
O
O
O
O
O
O
O
O
O
NH2
NH2
NH
NH
NH
NH2
NH2
(c)
(a)
N
(b)
HO
HO
N
H
H
NH
O
O
O
1
2
3
(d)
2
O
O
O
O
O
O
O
O
O O
O
OH
OH
O
O
O
O
O
O
N
(e)
N
N
OH
N
(f)
N
N
H
Mn OH2
O
H
H
N
O
O
N
N
OH
O
O
O
O
O
O
L
4
Mn-EDTA-EOB
a Reagents and conditions: (a) (Boc)2O, NaHCO3, 1,4-Dioxane, rt; (b) 4-Ethoxyaniline, DIPEA, HBTU, DMF, rt;
(c) TFA, CH2Cl2, 0 °C, 18 h; (d) tert-Butyl bromoacetate, DIPEA, KI, DMF, 110 °C, 4 h; (e) TFA, CH2Cl2, 0 °C, 22
h; (f) MnCl2·4H2O, 1M NaOH, pH 6.5.
Relaxivity and lipophilicity. To understand how the Mn-EDTA-EOB complex acts as a CA for
MRI, we compared the relaxivity of Mn-EDTA-EOB with that of Mn-EDTA, Mn-EDTA-BTA,
Mn-DPDP, Gd-DTPA, Gd-DTPA-EOB and Gd-BOPTA using a 1.5 T clinical MRI scanner
(Table 1). After conjugation with the EOB moiety, r1 relaxivity of Mn-EDTA-EOB increased
approximately 20% compared with that of the parent Mn-EDTA. The lipophilic moiety EOB
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might play an important role by slowing down the tumbling rate of Mn-EDTA-EOB and
contributing to the high relaxivity.30,32 In addition, Mn-EDTA-EOB displayed much higher
longitudinal relaxivity (r1) than the clinically approved Mn-DPDP; coordinated inner sphere
water might be responsible for this high relaxivity. Subsequently, we also measured relaxivity in
an aqueous solution of human serum albumin (HSA) because of the presence of the lipophilic
group in the structure of Mn-EDTA-EOB complex. Under this condition, the Mn-EDTA-EOB
complex exhibited higher r1 relaxivity value than Mn-DPDP, demonstrating an interaction
between HSA and Mn-EDTA-EOB chelate. When compared with Mn-EDTA-BTA, Mn-EDTA-
EOB did not show high r1 relaxivity as good as Mn-EDTA-BTA. Therefore, Mn-EDTA-EOB is
expected to show less contrast enhancement compared to Mn-EDTA-BTA. However, it is also
worth to note that overall contrast enhancement is dependent not on r1 relaxivity but on r1 /r2
ratio because r1 and r2 enhancements are competitive each other. To estimate the lipophilicity of
our synthesized complex, we also determined the octanol-water partitioning coefficient (log P).
The log P values are Mn-EDTA-EOB (-2.33), Mn-EDTA (-2.72), Mn-EDTA-BTA (-1.84), and
Mn-DPDP (-3.07) (Table 1). The log P value of Mn-EDTA-EOB is 3-fold lower than that of Mn-
EDTA-BTA but Mn-EDTA-EOB shows 2.45 and 5.5-fold higher lipophilicity than Mn-EDTA
and Mn-DPDP respectively.
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Table 1. Relaxivities and octanol-water partition coefficients data of Mn-EDTA-EOB, Mn-
EDTA, Mn-EDTA-BTA, Mn-DPDP, Gd-DTPA, Gd-BOPTA and Gd-DTPA-EOB in water, PBS
and HSA (64 MHz, 297 K).
r1 (mM-1 s-1)
r2 (mM-1 s-1)
r1/r2
Compound
logPoct/wat
ratiog
Water
PBS
HSAf
Water
PBS
HSAf
Mn-EDTA-
EOB
0.53(W)
0.37(H)
2.3 ± 0.10
2.4 ±0.11
-
6.3±0.29
4.3 ± 0.05 4.3 ± 0.04
16.9±0.71
−2.33
−2.72
Mn-EDTAb 1.9 ± 0.10
-
3.7 ± 0.10
-
-
0.51(W)
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Mn-EDTA-
BTAb
0.71(W)
0.43(H)
3.5 ± 0.10
1.6± 0.03
-
15.1±1.90 4.9 ± 0.10
-
34.5 ± 3.90
8.9± 0.2
−1.84
−3.07
Mn-
0.76(W)
0.58(H)
2.1± 0.1
5.2± 0.1
2.1± 0.1
2.5± 0.2
DPDPc,d
7
0.84(W)
0.97(H)
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9
Gd-DTPAb
3.3
-
-
4.3
-
3.9
-
-
4.4
-
-3.16
-2.23
Gd-
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4.0 ± 0.40
4.3 ± 1.0
0.93(W)
BOPTAe
Gd-DTPA-
EOBe
4.7 ± 0.40
-
-
5.1 ± 1.2
-
-
0.92(W)
-2.11
b Data obtained from ref 29. c, d Data obtained from refs 53 and 54. eData obtained from ref 32. f[HSA] = 0.67 mM in
water. g(W) for r1/r2 ratio in water, (H) for r1/r2 ratio in HSA solution.
obs
Interaction of Mn-EDTA-EOB complex with HSA. The R1 values of a diluted solution of
Mn-EDTA-EOB complex were measured to investigate the interaction of the lipophilic Mn-
EDTA-EOB complex with HSA. The measurements were performed as a function of protein
obs
concentration at the fixed frequency and temperature. R1 increases with increasing protein
concentrations because the fraction of bound complex, which is characterized by a slower
representational motion, increases. The binding constant (Ka) was determined according to a
previously published method.33 Figure 1 shows the results from proton T1 relaxation rate as a
function of Mn-EDTA-EOB concentration at a fixed HSA concentration. The data obtained for
Mn-EDTA-EOB offer a relatively sharp inflection point, which is an indication of high stability
constant. The binding constant (Ka) of Mn-EDTA-EOB (655 M-1) was much lower than that of
the protein binding agent MS-325 (6100 M-1), and there was no obvious brightness observed in
blood vessels after injection of Mn-EDTA-EOB to mice (data not shown).33 Weak protein
binding results in increased relaxivity, but does not appear to impact on blood pool contrast.
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Figure 1. Proton longitudinal paramagnetic relaxation rates of Mn-EDTA-EOB as a function of
Mn concentration in aqueous solution of HSA (0.67mM) at 64 MHz and 293 K, fitted using eq 1.
Transmetallation kinetics and pH stability. For safe in vivo application, the kinetic stability of
CAs is crucial. As the second most abundant transition metal in the human body, divalent zinc
(Zn2+) plays a critical role in many cellular processes and can displace more Mn2+ than other
endogenous metal ions such as Cu2+ and Ca2+. Thus, in this study, the stability of the Mn2+
complex was measured by determining the transmetallation kinetics of Zn2+ ions.
Transmetallation rates are frequently characterized by measuring the progress of normalized r1 or
r2 relaxivities as a function of time.28,29 The kinetic stability of Mn-EDTA-EOB was
characterized by a change in transverse relaxation rate, {ΔR2(t) = R2(t) - R2(0)} of complex
incubated with Zn2+ (10 equiv.) as a function of time. The kinetic stability of Mn-EDTA, Mn-
DPDP, Mn-EDTA-BTA, Gd-DTPA-EOB, and Gd-BOPTA was also tested for comparison
(Figure 2). As shown in Figure 2, Mn-EDTA-EOB shows much higher kinetic stability than
others for the 72-h experimental period. Although the manganese coordination with EDTA and
EDTA-EOB is similar, Mn-EDTA-EOB shows higher transmetallic stability than Mn-EDTA.
One possible explanation is that the steric hindrance induced by the EOB group located on the
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ethylene bridge might to have a favorable effect by reducing the accessibility of Zn2+ and/or by
rigidifying the structure of Mn-EDTA-EOB complex.34 Mn-EDTA-EOB shows higher kinetic
inertness [lower ΔR2(t) value] than the previously studied Mn2+-based liver-specific agent Mn-
EDTA-BTA, demonstrating that EOB has better rigidifying effect than BTA in terms of
transmetalation. Furthermore, the kinetic stability of Mn-EDTA-EOB was much higher than that
of clinically used Gd-based MRI CAs, such as Gd-DTPA-EOB and Gd-BOPTA. These results
indicate that Mn-EDTA-EOB has much higher kinetic inertness toward transmetallation by Zn2+.
In addition, stability of the Mn-EDTA-EOB was also measured at various pH range as a function
of the time. Little change in relaxivity was observed upon going from basic to acidic pH,
suggesting that Mn-EDTA-EOB is stable enough throughout a wide range of pH from 3 to 11
(see Figure S12). Thus, we expect that Mn-EDTA-EOB can be a good alternative to Gd-based
CAs, particularly for renally impaired populations where Gd usage carries additional risk. In
patients with low glomerular filtration rate (GFR), the Mn-chelate may circulate for extended
periods, and we assume that any slowly released Mn2+ may be excreted by the liver.
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Figure 2. Transmetallation of 1mM Mn-EDTA (▼), Mn-DPDP (X) Mn-EDTA-BTA (■), Gd-
DTPA-EOB (Ж), Gd-BOPTA (▲) and Mn-EDTA-EOB (●) by 10 mM Zn2+ plotted by ΔR2 as a
function of time 3 T and 293 K {ΔR2(t) = R2(t) – R2(0)}.
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Cell viability assay and histology. The cytotoxic effects of Mn-EDTA-EOB on healthy human
mammary epithelial cells (MCF-10A) and mouse normal liver cells (AML12) were evaluated by
performing in vitro cell viability assay (CCK). The cells were incubated with Mn-EDTA-EOB,
Gd-DTPA and Mn-DPDP complexes for 24 h. Here, Gd-DTPA was selected as a reference
complex because Gd-DTPA has a well-established cell viability data. The viability percentage of
the human mammary epithelial cells and mouse normal liver cells were retained above 82% and
88% for Mn-EDTA-EOB, whereas, clinically approved Mn-DPDP exhibit 63% and 79% cells
viability, when incubated with increasing concentrations up to 50 μM (Figure S14). Although
Mn-EDTA-EOB complex showed a decrease in cell viability at high concentration, the cell
viability data demonstrate that Mn-EDTA-EOB has negligible toxicity for MCF-10A and
AML12 cells at the dose required for MRI. In addition, to find out whether the injected Mn-
EDTA-EOB causes any impairment in vivo, comprehensive histological study of the liver tissue
section was carried out. H&E staining studies of the liver tissues did not show any apparent
damage in cellular structures after 2 h injection of Mn-EDTA-EOB compared to the liver tissue
without Mn-EDTA-EOB injection. No significant change in morphology was found up to 2 days
of injection (see Figure S15).
In vivo biodistribution and pharmacokinetics. The quantitative in vivo biodistribution of Mn-
EDTA-EOB chelate was evaluated by an inductively coupled plasma (ICP) spectrophotometer
(Figure 3). ICP data showed highest Mn2+ accumulation in the liver (around 26%) and
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correspondingly in the intestine, indicating that Mn-EDTA-EOB was eliminated through the
hepatobiliary route. For comparison, we selected Gd-DTPA-EOB complex, which is commercial
liver-specific MR CA because Mn-EDTA-EOB showed strong possibility as potential liver-
specific CA. Clinically used Gd-DTPA-EOB showed liver accumulation almost double within 30
min when compared with Mn-EDTA-EOB.32 Besides the liver, the kidneys also exhibit
comparatively high Mn accumulation, suggesting excretion via the renal pathway. The in vivo
biodistribution data demonstrated that Mn-EDTA-EOB is excreted through both hepatobiliary
and renal pathways. Although liver accumulation of Mn-EDTA-EOB is lower than that of Gd-
DTPA-EOB, Mn-EDTA-EOB displays similar biodistribution pattern to Gd-DTPA-EOB.30 It
should be noted that, during biodistribution studies the urine and feces were excluded from
bladder and intestine respectively. Also, the distribution half-life (td 1/2) and elimination half- life
(te 1/2) of Mn-EDTA-EOB complex were 11 min and 2.3 h respectively (see Figure S16). In
general, hepatobiliary excretion is relatively slower than renal clearance.
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Figure 3. Biodistribution patterns of Mn-EDTA-EOB (0.05 mmol Mn/g) in normal ICR mice
represented by Mn percentage in each tissue. Groups of mice (n = 4) were sacrificed at 10 min,
30 min, 2h and 24 h. Data are expressed as mean ± SD.
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In vivo MRI of normal mice. Six-week-old male ICR mice were used for in vivo T1-weighted
MRI after a bolus injection of Mn-EDTA-EOB through the tail vein (Figure 4). For comparison,
we again selected Gd-DTPA-EOB and Gd-BOTPA which are commercial liver-specific MR
CAs. The most distinctive MR feature of Mn-EDTA-EOB is that the contrast enhancement in the
liver was similar to Gd-based liver CAs, such as Gd-DTPA-EOB and Gd-BOTPA, and
gallbladder showed much higher enhancement after 60 min (Figure S17). Contrast-to-noise ratio
(CNR) graphs of Mn-EDTA-EOB shows considerable signal increases in the kidney and liver,
and correspondingly in the gallbladder. Together with the findings of biodistribution study, in
vivo MR images confirm that Mn-EDTA-EOB is excreted through both the liver and kidneys. In
the hepatobiliary pathway, Mn-EDTA-EOB is taken up by hepatocytes and eliminated via the
biliary trees. Because of the paramagnetic properties of Mn2+, Mn-EDTA-EOB causes T1
enhancement of the liver and biliary trees. This dual elimination pattern of Mn-EDTA-EOB is
similar to that of Gd-based liver-specific agents Gd-DTPA-EOB and Gd-BOTPA.30,35
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Figure 4. In vivo T1-weighted spin echo (SE) MR images of normal ICR mice obtained after tail
vein injection of Mn-EDTA-EOB (0.05 μmol Mn/g); G.B., gallbladder.
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In vivo MRI characterization of liver tumor. To investigate the clinical efficacy of Mn-EDTA-
EOB chelate as a liver-specific CA, we performed in vivo MRI of liver tumor using an Huh-7
xenograft mouse tumor model after bolus injection of Mn-EDTA-EOB through the tail vein
(Figure 5a). The liver tumor location and size were confirmed with T2-weighted MR images
before Mn-EDTA-EOB injection. Immediately after intravenous injection of Mn-EDTA-EOB,
tumor tissues can be distinguished from the normal liver tissues owing to the greater signal
intensity of normal liver than tumor tissues in T1-weighted MR images (Figure 5a). Unlike
extracellular fluid (ECF) agents, liver-specific hepatobiliary MRI CAs can be transported into
normal hepatocytes.36,37 The transportation of liver-specific MRI CAs into hepatocytes involves
transport systems at the sinusoidal and canalicular membranes of hepatocytes.38 The liver-
specific CAs enter normal hepatocytes mostly through organic anion-transporting polypeptide
transporters (OATPs).39 However, OATPs are considerably reduced in HCC cells; therefore,
HCC cells do not take up or excrete lipophilic agents and appear hypointense to the normal
liver.40,41
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In addition, CNR shows marked differences in the uptake of Mn-EDTA-EOB between normal
hepatocytes and tumor cells, indicating a substantial difference in MR signal enhancement
(Figure 5b). Moreover, color mapping images specify the normal (peach color) and tumor
(brown color) liver regions after 90 min injection of Mn-EDTA-EOB when compared with pre-
images (Figure 5c). Furthermore, we obtained an image histogram that shows the number
of pixels at each different intensity value. Before injection of Mn-EDTA-EOB, the histogram
showed insignificant signal intensity difference between normal and tumor regions. However,
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after Mn-EDTA-EOB injection, the histogram showed a considerably higher signal intensity
value of normal liver than that of tumor tissues, revealing the significantly enhanced normal
hepatocytes (Figure 5d). Therefore, Mn-EDTA-EOB demonstrated remarkable clinical potential
to detect liver tumors by distinguishing normal liver tissues from tumor tissues.
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Figure 5. Axial T2-weighted and T1-weighted MR images of Huh-7 xenograft mice (a) before
and after injection of Mn-EDTA-EOB (0.05 mmol Mn/kg). Yellow arrow indicates the liver
tumor lesion. (b) CNR profiles of normal (red circles) and tumor (blue squares) liver tissue as a
function of time. CNR was measured in axial T1-weighted images. (c) Color mapping images of
pre and post 90 min injection of Mn-EDTA-EOB. (d) Histogram of the pre and post 90 min axial
images as signal intensity values. The signal intensity of tumor and normal liver regions was
indicated by blue and red bars respectively. The vertical line of each distribution represents the
average value. Statistical significance of the differences between the intensity of normal and
tumor tissue was analyzed using the Mann-Whitney U test with SPSS software (**, P ≤ 0.01; Z
= -5.547 for b; Z = -27.419 for d).
Molecular docking simulation. The biodistribution pattern of Mn-EDTA-EOB complex is
similar to that of Gd-DTPA-EOB and there is ample evidence that hepatic uptake of Gd-DTPA-
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EOB is mediated by liver specific-uptake transport proteins such as OATPs and/or
Na+/taurocholate cotransporting polypeptide (NTCP).42,43 We hypothesized that hepatic uptake
of Mn-DTPA-EOB can be mediate by OATPs and/or NTCP due to the similar biodistribution
pattern with Gd-DTPA-EOB and presence of EOB moiety in the compound structure. In many
studies, molecular docking has been used to identify the effective binding sites and affinities.44,45
Therefore, we performed computational modeling studies to identify the favorable transporter
system for the uptake of Mn-EDTA-EOB complex into the liver.45 Homology modeling
approach was introduced to estimate the three-dimensional structures of OATPs and NTCP
because of the lack of crystal structures of these proteins. Subsequently, protein-ligand docking
simulations were performed.
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Figure 6 shows the possible interactions between different transporter systems and Mn-EDTA-
EOB. Among the different transporter systems, the results of docking simulation study
demonstrated that OATP1b3 and OATP2b1 are more favorable than other transporter systems.
Mn-EDTA-EOB forms several hydrogen bonds with OATP1b3 transporter, and K49 in
OATP1b3 forms a salt bridge with Mn-EDTA-EOB complex. OATP2b1 forms hydrogen bonds
with Mn-EDTA-EOB chelate, and K70 in OATP2b1 forms several salt bridges with Mn
complex. F156 of OATP2b1 also forms pi-pi stacking with the phenyl ring of Mn complex. The
docking scores, which were calculated using Schrödinger suite software, between Mn-EDTA-
EOB and transport proteins were -11.58 kcal/mol for OATP1b3 and -11.12 kcal/mol for
OATP2b1 (Table 2). However, OATP1b1 and NTCP only form hydrogen bonds and/or salt
bridges with Mn-EDTA-EOB complex. The docking score between Mn-EDTA-EOB and
OATP1b1 was -9.36 kcal/mol and that between Mn-EDTA-EOB and NTCP was -10.67 kcal/mol
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(Table 2). Therefore, the docking simulation studies demonstrate that Mn-EDTA-EOB uses
OATP1b3 and OATP2b1 as the major gateway for hepatic uptake.
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For comparison, computational docking studies of Gd-DTPA-EOB, EDTA-EOB and Mn-
EDTA also performed and are shown in Table 2 and Figure S18-S20. Mn-EDTA-EOB shows
much higher interaction with different transporters, when compared with EDTA-EOB and Mn-
EDTA. One of the major modes of transport is the OATP1 series. Although 1b1 and 1b3 of
OATP are expressed in normal liver tissue, the expression of OATP1b1 and OATP1b3 decreases
in liver tumors according to the degree of differentiation of tissues.46 This pattern is in good
agreement with in vivo MR image (Figure 5).
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Figure 6. Schematic representations of variable interactions between Mn-EDTA-EOB and (a)
OATP1b1, (b) OATP1b3, (c) OATP2b1 (PDB ID: 2GFP, gray), and (d) NTCP (PDB ID: 3ZUY,
gray). These images show the amino acids within 5 Å around the Mn-EDTA-EOB. The yellow
line represents hydrogen bond, the pink line represents salt bridge, the green represents pi-cation
interaction, and the cyan represents pi-pi stacking interaction.
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Table 2. Docking scores of the Mn-EDTA-EOB, EDTA-EOB, Mn-EDTA and the commercial
agent Gd-DTPA-EOB with liver specific-organic anion transporter proteins.
OATP1b3
-11.58
-11.54
-9.24
OATP1b1
-9.36
OATP2b1
-11.12
-12.38
-9.83
NTCP
-10.67
Mn-EDTA-EOB
Gd-DTPA-EOB
EDTA-EOB
-13.25
-8.99
-12.78
-13.25
Mn-EDTA
-8.75
-9.83
-8.07
-9.61
(kcal/mol)
CONCLUSION
In the present study, we synthesized a novel Mn-EDTA complex with high kinetic inertness by
combining a lipophilic EOB moiety and studied its possible diagnostic efficacy as a liver-
specific MRI CA. It showed higher r1 relaxivity (2.3 mM−1 s−1) than clinically approved Mn-
DPDP (1.6 mM−1 s−1) at 1.5 T. The kinetic inertness of Mn-EDTA-EOB chelate is much higher
than that of Gd-based liver-specific MRI CAs (Gd-DTPA-EOB and Gd-BOPTA) and previously
reported Mn-based liver-specific CAs (Mn-DPDP and Mn-EDTA-BTA). The biodistribution
pattern and in vivo MR images showed that Mn-EDTA-EOB is excreted through both
hepatobiliary and renal pathways. The molecular docking simulation study suggests that Mn-
EDTA-EOB might use OATP1b3 and OATP2b1 transport systems as the major gateway for
hepatic uptake. Finally, in a liver tumor model, Mn-EDTA-EOB demonstrated significant
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improvement in tumor detection and characterization, suggesting that this new chelate can be a
prominent MRI agent for liver cancer, particularly in patients with compromised renal function.
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EXPERIMANTAL PROCEDURES
General Remarks. All reagents were purchased from Tokyo Chemical Industry (Tokyo, Japan),
Sigma-Aldrich (St. Louis, MO, USA), and/or Alfa-Aesar (Ward Hill, MA, USA) and used
without additional purifications. Solvents were dried and purified using standard methods.
Deionized (DI) water was used for all experimental procedures.1H NMR spectrums were
measured on a 500 MHz (Bruker Advance) at the Kyungpook National University (KNU).
Tetramethyl silane (TMS) was used as an interior standard for chemical shifts. Elemental
analyses (EA) were accomplished in KNU. The detailed methods for High-resolution fast atom
bombardment mass spectrums (HR-FAB-MS) and High-pressure liquid chromatography (HPLC)
are given elsewhere (See reference 26). Electrospray ionization mass spectrometry (ESI-MS)
was obtained by using a Q Exactive quadrupole Orbitrap mass spectrometer (Thermo Fisher
Scientific Inc., Rockford, IL, USA) at department of chemistry, KNU. Methods used for purity
tests were as follows: Eluent A, 10 mM ammonium acetate in water; B, 10 mM ammonium
acetate in ACN; gradient, 5% B to 70% B in 25 min, 70% B to 100% B in 5 min, 100% B in 10
min; flow rate 12 mL/min. Purity of the synthesized compounds were confirmed by elemental
analysis or a reverse-phase HPLC with UV−vis detection at 254 nm and purity of the final
complex was 95% or higher. No unexpected or unusual high safety hazards were encountered
during experimental procedures.
Synthesis and Characterization. 2, 3-Bis-tert-butoxycarbonylaminopropionic Acid (1). Title
compound was synthesized according to a published literature with slight modifications.29
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[2-tert-Butoxycarbonylamino-2-(4-ethoxy-phenylcarbamoyl)-ethyl]-carbamic acid tert-butyl
ester (2). Compound 1 (2 g, 6.57 mmol), N, N-diisopropylethylamine (1.6 mL, 13.15 mmol),
HOBt (1.25 g, 8.21 mmol) and HBTU (3.73g, 9.85 mmol) were dissolved in DMF. 4-
Ethoxyaniline (1.13g, 8.21 mmol) was slowly added and stirred for 18 h at room temperature.
Resulting mixture was diluted with DI water and extracted with ethyl acetate (3×20 mL).
Organic layer was washed with brine and dried over MgSO4. Crude compound was recrystallized
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with dichloromethane and pure product was collected as a white solid. Yield: 1.9 g (69%). H
NMR (MeOD-d4): δ = 9.77 (s, 1H, NH), 7.47 (s, 2H, NH) 6.86−6.79 (dd, 4H, CH), 4.41 (t, 1H,
CH), 3.97 (dd, 2H, CH2), 1.39 (s, 18H, CH3), 1.32 (t, 3H, CH3), 1.29 (d, 2H, CH2). Anal. Calcd
for C21H33N3O6: C, 59.56; H, 7.85; N, 9.92. Found: C, 59.30; H, 7.95; N, 9.90. HR-FAB-MS
(m/z) for C21H33N3O6: calcd, 423.2369 [M] + and 424.2448 [M + H] +; found, 423.2372 [M] + and
424.2445 [M + H] +.
2, 3-Diamino-N-(4-ethoxy-phenyl)-propionamide (3). Trifluoracetic acid (TFA) was carefully
added into the solution of compound 2 (1.5 g, 3.54 mmol) in dichloromethane (CH2Cl2) at 0 °C.
For 18 h, the solution was stirred to consume starting materials. Solvent was removed and ethyl
ether was added to obtained precipitate. Solid appears was filtered and washed with ethyl ether
several times. After vacuum dried pure product was collected as pale-yellow solid. Yield: 0.75 g
(95%). 1H NMR (MeOD-d4): δ = 7.54 (d, 2H, CH) 6.89 (d, 2H, CH), 4.32 (t, 1H, CH), 4.02 (dd,
2H, CH2), 1.37 (t, 3H, CH3), 1.18 (d, 2H, CH2). Anal. Calcd for C11H19N3O3·4CF3CO2H: C,
32.72; H, 3.32; N, 6.03. Found: C, 33.06; H, 3.29; N, 5.93. HR-FAB-MS (m/z) for C11H18N3O2:
calcd, 224.1399 [M + H] +; found, 224.1402 [M + H] +.
{[2-(Bis-tert-butoxycarbonylmethyl-amino)-1-(4-ethoxy-phenylcarbamoyl)-ethyl]-tert-
butoxycarbonylmethyl-amino}-acetic acid tert-butyl ester (4). Compound 3 (2 g, 8.92 mmol),
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diisopropylethylamine (6.92 mL, 53.57 mmol) and potassium iodide (2.96 g, 17.84 mmol) were
dissolved in DMF. tert-Butyl bromoacetate (10.43 mL, 53.57 mmol) was added dropwise and
heated to 110 °C for 4 h. Solvent was removed and residue was partitioned between ethyl acetate
and 10% NaHCO3 solution. Organic layer was collected, washed with brine and dried over
Na2SO4. After evaporation crude product was collected and purified by column chromatography
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(silica, hexanes/EtOAc, 9:1). Yield: 5.4 g (88%). H NMR (DMSO-d6): δ = 10.23 (s, 1H, NH),
7.47 (d, 2H, CH) 6.88 (d, 2H, CH), 3.98 (dd, 2H, CH2), 3.61 (t, 1H, CH), 3.57-3.40 (m, 8H, CH2),
3.18-2.89 (dd, 2H, CH2), 1.40 (s, 36H, CH3), 1.30 (t, 3H, CH3). Anal. Calcd for
C35H57N3O10·2H2O: C, 58.72; H, 8.59; N, 5.87. Found: C, 58.35; H, 8.73; N, 5.83. HR-FAB-MS
(m/z) for C35H58N3O10: calcd, 680.4122 [M + H]+; found, 680.4127 [M + H]+.
{[2-(Bis-carboxymethyl-amino)-1-(4-ethoxy-phenylcarbamoyl)-ethyl]-carboxymethyl
amino}-acetic acid (L). Compound 4 (1.5 g, 2.20 mmol) was dissolved in a mixture of
trifluoracetic acid and dichloromethane using ice bath. Resulting solution was stirred until
starting materials was disappeared. Ether was added to obtained precipitate after removing
1
solvent. Then filttred and dried to get pure product as off-white solid. Yield: 0.96 g (96%). H
NMR (MeOD-d4): δ = 7.48 (d, 2H, CH) 6.87 (d, 2H, CH), 4.09 (d, 4H, CH2), 3.98 (dd, 2H, CH2),
3. 57-3.56 (m, 6H, CH2), 1.46 (t, 3H, CH3). Anal. Calcd for C19H25N3O10·1/2CF3CO2H: C, 47.20;
H, 5.07; N, 8.31. Found: C, 46.85; H, 5.15; N, 8.28. HR-FAB-MS (m/z) for C19H26N3O10: calcd,
456.1618 [M + H] +; found, 456.1621 [M + H] +.
[Mn (EDTA-EOB) (H2O)]2-. Ligand L (0.6 g, 1.3 mmol) was dissolved in DI water and pH of
the solution was adjusted to 6.5 by carefully addition of NaOH (1M). MnCl2·4H2O (0.26 g, 1.3
mmol) was slowly added and resulting solution was stirred for three days at RT. During this time
pH of solution was readjusted for several times. After lyophilization crude product was collected
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as solid and extra purification was performed by preparative HPLC and purity was also
confirmed (see Figure S10). Yield: 0.38g (58%). ESI-MS (m/z) for C19H22MnN3O10: calcd,
507.0686 [M+H] +; found, 507.0682 [M+H] +.
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Relaxivity. Relaxivities were measured with 1.5 T GE unit (GE Healthcare). T1 measurements
were carried out at 35 different inversion times (TIs) range between 50 and 1750 ms using an
inversion recovery technique. In case of T2, the CPMG (Carr−Purcell−Meiboon−Gill) pulse
sequence was used at 34 different echo times (TEs) range from 10 to 1900 ms. The nonlinear
least-squares fit of the average pixel were used to estimate T1 and T2 relaxation. Then, r1 and r2
relaxivities were computed by the linear fit of T1 and T2 relaxation time for each concentration
(0.0625, 0.125, 0.25, 0.5, and 1 mM).
Octanol-Water Partition Coefficients. This experiment was accomplished according to a
published literature method.47 1 mg sample of Mn-EDTA-EOB complex was dissolved in 2 mL
of water and 1-octanol mixture (1:1 v/v). Resulting mixture was shaken for a second, then the
vial was gently mixed on a rotator for 48 h. The mixture was then placed on the desk and
allowed to be divided into two layers for 24 h at room temperature. Mn concentrations of each
layer were measured by inductively coupled plasma mass spectrometry (ICP-MS). Octanol-water
partition coefficients were computed using the equation log P = log(Co/Cw), where log P is the
common logarithm of the partition coefficient, Co and Cw are the concentration of Mn in the 1-
octanol layer and water layer, respectively.
Transmetallation Kinetics. This experiment was directed according to the published procedure
with trivial modifications.28,29 50 μL of a 100 mM MES [2-(N-morpholino) ethane sulfonic acid]
buffered solution (pH 6) of ZnCl2 was added to a 450 μL of 1.25 mM buffered solution of Mn-
EDTA-EOB complex. The final concentration we use for this experiment was 1 mM solution of
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Mn-EDTA-EOB complex and 10 mM ZnCl2. The mixture was shaken for a while then instantly
used for measuring solvent T2. For comparison purpose Mn-EDTA, Mn-EDTA-BTA, Mn-DPDP
(Teslascan), Gd-DTPA-EOB (Primovist) and Gd-BOPTA (MultiHance) were also studied. The
measurements were conducted on a 3 T system (Magnetom Tim Trio, Siemens, Germany) for
72 hours at room temperature. The diagram was plotted using the equation {ΔR2(t) = R2(t) −
R2(0)} as a function of time. N=3 for each complex and average value was used for calculation.
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Determination of Binding Constants. The binding affinity of Mn-EDTA-EOB chelate with
HSA was measured by method in literatures.33,48 The nonlinear rise of the 1H relaxation rates was
fitted using eq 1, where N is the number of independent interaction sites (N was set to 1), p0 is
c
f
the concentration of the HSA, s0 is the paramagnetic complex concentration, and r1 and r1 are
the relaxivities of the complex HSA-contrast agent and of the free contrast agent, respectively.
표푏푠
푅1푝
= 1000
1
푓
0
푐
푓
(
)
× 푟1 × 푠 + (푟1 ― 푟1)
{
2
푁 × 푝 + 푠 + 퐾푎 2 ― 4 × 푁 × 푠0 × 푝0
0
0
―1
0
0
―1
(
(
)
(
(
)
)
푁 × 푝 + 푠 + 퐾푎
―
)
}
(1)
Kinetic stability in various pH regions. The stability was given in the evolution of the water
proton relaxation rate (R1) for Mn-EDTA-EOB in various buffer solution at different pH ranges
(pH: 1,3,5,7,9 and 11) as a function of time.49 The measurements were performed on a 3 T whole
body system (Magnetom Tim Trio, Simens, Germany) at room temperature for 12 days.
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Cell Culture. The culture medium contained of 1% (v/v) penicillin−streptomycin, 10% (v/v)
fetal bovine serum (FBS), and Dulbecco’s Modified Eagle Medium/Nutrient Mixture F-12
(DMEM/F12, Gibco Invitrogen, Carlsbad, CA).
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Cell preparation for MR imaging. Mouse normal liver cells (AML12) and human mammary
epithelial cells (MCF-10A) were plated at a density of 2 X 105 in 35 mm dishes. The DMEM
growth medium was removed, and the cells were incubated with Mn-EDTA-EOB (0, 5 and 50
μM) in DMEM/F-12 growth media for 24 h. The cells were washed with phosphate buffered
saline (PBS) and harvested. Media with trypsin-EDTA was removed and cells were resuspended
with DMEM/F-12 growth media.
Cell Viability Assay. Human mammary epithelial cell line (MCF-10A) and mouse normal liver
cell line (AML12) were purchased from ATCC (Manassas, Virginia) and plated in 96-well plates
at a density of 1 × 104. The DMEM/F-12 growth medium were removed and Mn-EDTA-EOB,
Mn-DPDP and/or Gd-DTPA chelates were incubated with cells in DMEM/F-12 serum-reduced
medium for 24 h. The cell viability of Mn-DPDP with various Mn2+ concentration was provided
as control. Using the CCK-8 kit (Dojindo, Sunnyvale, CA), cell viability was computed
according to the manufacturer’s protocol. Briefly, to each well CCK-8 solution (10 μL) was
added then the samples were incubated for 2 h and absorbance of samples were detected at 450
nm.
Pharmacokinetic study. Pharmacokinetic studies were conducted by following previously
published procedure with slight modification.50 Blood plasma was assessed on 5-week-old male
BALB/c mice (28-30 g). Blood samples (total 60 μL) were pooled from three mice (20 μL from
each mice) for one group and four group were used for study. Blood samples were collected
from the tail vein before injection and at 10, 30 min, 1, 3, 6, 9 and 12 hour after injection of Mn-
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EDTA-EOB at a dose of 0.05 mmol/kg. The collected samples were immediatly mixed with
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heparin solution and centrifuged at 5000 rpm at 4 C for 10 min to obtain plasma. The plasma
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were treated with 70 % nitric acid (HNO3) and 30% hydrogen peroxide (H2O2) at 180 °C for 3 h.
The manganese concentration of the treated sample was measured by an inductively coupled
plasma spectrophotometer (NexION 300X, PerkinElmer, USA). The recognition limit of this
process is 1 ppb. Blood plasma pharmacokinetics were estimated by plotting the Mn
concentration as a function of time and fitting the decay to a biexponential function using
MATLAB curve fitting tool. The distribution and elimination half-lives (td 1/2, te 1/2) were
estimated, respectively.
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H&E staining. For histological evaluation, portions of liver collected on 2 hour and 2 day after
injection of Mn-EDTA-EOB (0.05 mmol/kg) were fixed in 4 % paraformaldehyde buffer
solution for one and a half day and then embedded in paraffin blocks. Tissue sections 3 μm thick
were stained with hematoxylin and eosin (H&E).51
In vitro MR Imaging. In vitro MR images were obtained using a 3 T MR scanner (GE
Healthcare, Milwaukee, WI, USA). The imaging parameters for T1-weighted image were as
follows: repetition time (TR) = 300 ms; echo time (TE) = 9 ms; 26 mm field of view (FOV); 192
X 192 matrix size; 13.0 mm slice thickness; number of acquisition (NEX) = 15.
In Vivo MR Imaging. All animal trials were permitted by Kyungpook National University
Animal Care Committee and tests were performed in agreement with their rules. For MRI studies,
all animal protocols were same as the protocols given previously (See reference 29). MR
measurements were performed with a 1.5 T MRI (GE healthcare) and imaging protocols
including sequence parameters were same as the methods given previously (See reference 29).
The color-mapped images at maximum enhanced time (90 min) and the histograms of the normal
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and tumor liver region were obtained by using Python 3.6. All scripts were composed by using
the python library Matplotlib.
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Liver Tumor Model. An orthotopic model of liver cancer was used by the approval of the
Institutional Animal Care and Committee of Daegu-Gyeongbuk Medical Innovation Foundation
(DGMIF). Five-week-old male nude mice (BALB/c nu/nu) were obtained from Central Lab
Animal Inc. (Seoul, Korea). The Huh-7 cell line was gained from PerkinElmer Inc. and cells (5 ×
106 cells in 50 μL of DMEM) were inoculated in the liver of the mice.
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Image Analysis. The program Image J (NIH, USA) was used for measurement of signal
intensity values for specific regions of interest (ROI). The CNR (contrast-to-noise ratio) was
estimated by means of eq 2, where SNR is the signal-to-noise ratio.
CNR = SNRpost – SNRpre
(2)
Biodistribution study. After intravenously injection of Mn-EDTA-EOB (0.05 mmol/kg) to six-
week-old normal male mice (ICR; 25-30 g) through tail veins, the mice were anesthetized and
sacrificed at 4 different time points (10 min, 30 min, 2 h and 24 h after injection). Blood and
various organs were collected and treated with 70 % nitric acid (HNO3) and 30% hydrogen
peroxide (H2O2) at 180 °C for 3 h. The manganese concentration of the treated sample was
measured by an inductively coupled plasma spectrophotometer (ICP-spectrophotometer, Optima
7300DV, PerkinElmer, USA). The recognition limit of this process is 0.01 ppm.52
Statistical analysis. Data are presented as mean ± standard error if there is no other description.
Statistical analysis was performed using the Mann-Whitney U test with SPSS software. Curve
fits were done using MATLAB curve fitting tool.
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Molecular Modeling. Three-dimensional structures of OATP1B1, OATP1B3, OATP2B1 and
NTCP were predicted using homology modeling method. Crystal structure of the multidrug
transporter EmrD (PDB ID: 2GFP) and crystal Structure of a bacterial homologue of the bile
acid sodium symporter Asbt (PDB ID: 3ZUY) were selected as template structures for OATPs
and NTCP homology modeling, respectively. Three-dimensional structures of the four proteins
were constructed by Prime [55] homology modeling application. After that, Prime [55] structure
optimization tool was performed to optimize the structures under implicit solvent and membrane
environments. Protein-ligand docking calculations were performed using Glide (Grid-based
Ligand Docking with Energetics) [56] of the Schrödinger suite. Glide searches for favorable
interactions between small molecules and a receptor protein. In Glide, molecules were described
using OPLS 2005 force filed and the SP (standard precision) mode of Glide was used allowing
flexible ligand sampling. Delta G scores of the docked ligands were calculated by empirical
scoring function called Glide score.
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AUTHOR INFORMATION
Corresponding Author
*Y.C.: E-mail, ychang@knu.ac.kr. Phone, (+)82-53-420-5471.
ORCID
Yongmin Chang: 0000-0002-0585-8714
Md. Kamrul Islam: 0000-0001-8411-4766
Author Contributions
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